Continued support is requested to test the hypothesis that CD98 is important in the proliferation and function of cells in injured blood vessels. The applicant will examine the effect of CD98 deletion on the response to arterial injury in vivo and on vascular cell proliferation and sun/ival ex vivo and in vitro. He will use CD98 mutants that specifically couple to integrins or amino acid transporters to examine the relative importance of each in vascular cell proliferation and survival. If integrins are implicated, he will examine the role of CD98 in integrin-dependent signals (e.g. pp125FAK, ERK1/2, PIS Kinase) and conversely will identify the relevant integrins. Finally in collaboration with Core Unit A (Ruggeri) and Project 3 (Ruf) he will examine the effect of CD98 deletion in smooth muscle cells and myeloid cells in the generation of procoagulant activity in vivo and in vitro.
A second Aim will test the hypothesis that shear stress results in localized activation of Protein Kinase A in endothelial cells and that this localized activation directs endothelial cell alignment and migration by phosphorylating integrin aA and other substrates to modify the activity of Rho GTPases. He will use a microfluidic platform in combination with FRET-based biosensors to examine changes in PKA activity, in real time, in migrating vascular cells and in endothelial cells in response to shear stress. A similar approach will be used to assess responses of Rho GTPases using their biosensors and cross correlation and computational multiplexing will be used to examine the spatiotemporal relationship of Rho GTPase activity with protrusion and with PKA activity. He will inhibit PKA activity in a type-specific manner by using type specific AKAPs joined to mitochondrial targeting sequences to sequester either Type I or Type II PKA and examine effects on Rho GTPases, alignment, and cell migration. Finally, he will identify PKA substrates that are important in regulating Rho GTPase activity. These studies, using cutting edge methodologies, will provide new insight Into the mechanisms that regulate the response of vascular cells to vascular injury and will provide detailed mechanistic insight into the control of endothelial cell migration into wounds.

Public Health Relevance

This project proposes to analyze the role of adhesive signaling in the response of vascular cells to vessel injury. The analysis of these signaling events may identify novel means of stabilizing vessel integrity to promote thromboresistance. Thus, these studies are relevant to the prevention of the complications of cardiovascular disease and of vascular interventions

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Program Projects (P01)
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Heart, Lung, and Blood Initial Review Group (HLBP)
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Scripps Research Institute
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Langer, Florian; Ruf, Wolfram (2014) Synergies of phosphatidylserine and protein disulfide isomerase in tissue factor activation. Thromb Haemost 111:590-7
von Drygalski, Annette; Bhat, Vikas; Gale, Andrew J et al. (2014) An engineered factor Va prevents bleeding induced by anticoagulant wt activated protein C. PLoS One 9:e104304
Winkler, Ethan A; Sengillo, Jesse D; Sagare, Abhay P et al. (2014) Blood-spinal cord barrier disruption contributes to early motor-neuron degeneration in ALS-model mice. Proc Natl Acad Sci U S A 111:E1035-42
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von Drygalski, Annette; Furlan-Freguia, Christian; Ruf, Wolfram et al. (2013) Organ-specific protection against lipopolysaccharide-induced vascular leak is dependent on the endothelial protein C receptor. Arterioscler Thromb Vasc Biol 33:769-76
Wang, Yaoming; Sinha, Ranjeet Kumar; Mosnier, Laurent O et al. (2013) Neurotoxicity of the anticoagulant-selective E149A-activated protein C variant after focal ischemic stroke in mice. Blood Cells Mol Dis 51:104-8
Liu, Yani; Davidson, Brian P; Yue, Qi et al. (2013) Molecular imaging of inflammation and platelet adhesion in advanced atherosclerosis effects of antioxidant therapy with NADPH oxidase inhibition. Circ Cardiovasc Imaging 6:74-82
Burnier, Laurent; Fernandez, Jose A; Griffin, John H (2013) Antibody SPC-54 provides acute in vivo blockage of the murine protein C system. Blood Cells Mol Dis 50:252-8

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